CN109207473A - A kind of cervical cell lytic reagent box and cleavage method - Google Patents
A kind of cervical cell lytic reagent box and cleavage method Download PDFInfo
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- CN109207473A CN109207473A CN201811153649.1A CN201811153649A CN109207473A CN 109207473 A CN109207473 A CN 109207473A CN 201811153649 A CN201811153649 A CN 201811153649A CN 109207473 A CN109207473 A CN 109207473A
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
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- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
Abstract
The present invention provides a kind of cervical cell lytic reagent box and cell lysing methods, belong to cell lysis techniques field, and the kit includes lysis buffer and Proteinase K；The lysis buffer includes the component of following concentration: the Tris-HCl of 10~500mM, the EDTA of 1~80mM, 0.05~2wt%SDS and 100~250mM NaCl.The cell lysing methods are uniformly mixed the following steps are included: after PBS buffer solution cleaning cervical cell sample liquid with lysis buffer, and Proteinase K mixing, 53~58 DEG C of 10~14h of digestion are added；Then it is vortexed after 30~60s of concussion, 53~58 DEG C are continued 1.5~2.5h of digestion and complete cell cracking；The kit and cleavage method can cervical cell sufficiently in lysate sample, the DNA of high concentration and high quality is dissociateed, conducive to the extraction and amplification of subsequent DNA.
Description
Technical field
The invention belongs to cell lysis techniques field more particularly to a kind of cervical cell lytic reagent boxes and cleavage method.
Background technique
Cervical carcinoma is to threaten one of the common cancer of women's health, effective screening and early diagnosis, can be significant
Reduce the incidence and the death rate of cervical carcinoma.Uterine neck ThinPrep liquid-based cytology test (TCT) has become cervical carcinoma screening and thin
Born of the same parents learn one of the common method of diagnosis.TCT detection is that a kind of be stored in cast-off cells saves in liquid, and passes through dedicated film-making
Instrument is attached to cell is evenly dispersed on glass slide, carries out the technology of dyeing observation.When clinical acquisitions sample, application is special
Uterine neck bush be inserted into cervix opening, the brush head of Uterine neck bush is placed in equipped with thin by the Exfoliative cells of swipe cervix opening and cervical canal
It is rinsed in the bottle of born of the same parents' preservation liquid, obtains cell sample.Later, the cell in liquid is saved through mixing, filtering, transfer,
It finally attaches on wave carrier piece, carries out cytodiagnosis after dyeing is fixed.But the uterine neck due to testing staff's subjective factor etc.
ThinPrep liquid-based cytology test the case where there are still missing inspections, therefore, the diagnosis of cervical carcinoma at present can not depend merely on cytology inspection
It looks into；In addition, what oncotherapy at this stage sought is accurate personalized treatment, it is necessary to be carried out from genetics angle to subject
The detection of cervical cancer-related genes.
And the premise for extracting the cell DNA of high concentration high quality is abundant lytic cell, cell cracking can be made by machinery
It is realized with, chemical action and enzyme effect.In TCT detection process, saving liquid can be good at saving cervical cell, Er Qieshou
Inspection person bears the optimal sample that pain is small, and undoubtedly cervical cancer gene detects.Contain in TCT detection cervix cell preservation solution
Cell component and acellular components；Cell component includes epithelial cell and non-epithelial cell, epithelial cell include squamous cell and
Endocervical cells, non-epithelial cell include haemocyte, such as red blood cell and interstitial cell.Acellular components include inherently at
Point, such as mucus, bacterium and pollutant, such as drug crystallization, fibre object, fungi.Therefore, it saves in liquid in addition to epithelium
Cell, other compositions are distracter.In addition, saving the chemical component in liquid also containing fixed cell, this kind of chemical component meeting
It crosslinks DNA with protein, influences the release of DNA, therefore saving liquid itself also is the interference for extracting cervical cell DNA
?.
Since the distracter in sample is numerous, existing commercial kit at all can not save DNA from uterine neck TCT
It sufficiently being dissociateed in liquid, the DNA mass and concentration thus extracted be not up to standard, subsequent amplification experiment can not be carried out, it is even more impossible to
Obtain accurate genetic test result.
Summary of the invention
In view of this, the present invention provide it is a kind of for uterine neck ThinPrep liquid-based cytology test save liquid sample uterine neck it is thin
Cellular lysate kit and cleavage method；The kit and cleavage method can cervical cell sufficiently in lysate sample, dissociation
The DNA of high concentration and high quality out, conducive to the extraction and amplification of subsequent DNA.
To achieve the goals above, the present invention provides a kind of cervical cell lytic reagent box, including lysis buffer and
Proteinase K；The lysis buffer takes water as a solvent, the component including following concentration: the Tris-HCl of 10~500mM, 1~
EDTA, the 0.05~2wt%SDS and 100~250mM NaCl of 80mM.
Preferably, the lysis buffer takes water as a solvent, the component including following concentration: the Tris- of 50~400mM
HCl, the EDTA of 5~60mM, 0.1~1wt%SDS and 150~220mM NaCl.
Preferably, the lysis buffer takes water as a solvent, the component including following concentration: the Tris-HCl of 100mM,
EDTA, 0.1wt%SDS and 200mM NaCl of 50mM.
Preferably, the pH value of the Tris-HCl is 8.0~8.3.
The present invention also provides the methods of kit cracking cervical cell, comprising the following steps:
1) PBS buffer solution cleans cervical cell sample liquid, is separated by solid-liquid separation, and collects solid phase components；
2) solid phase components are mixed with lysis buffer, obtains mixed liquor；
3) mixed liquor is mixed with Proteinase K Solution, carries out first stage digestion, the first stage digestion
Temperature is 53~58 DEG C, and the time is 10~14h, obtains digestive juice；
4) digestive juice is vortexed after shaking 30~60s, continues second stage digestion, the second stage disappears
The temperature of change is 53~58 DEG C, 1.5~2.5h of time；
The cervical cell sample liquid derives from the clinic detected preservation liquid containing cervical cell of uterine neck TCT.
Preferably, the PBS buffer solution and the volume ratio of cervical cell sample liquid are 1~5:1.
Preferably, the volume of lysis buffer described in step 2) and cervical cell sample liquid described in step 1)
Than for (1~5): (8~12).
Preferably, the concentration of the Proteinase K Solution is 0.1~2mg/ml.
Preferably, the volume ratio of lysis buffer described in step 2) and the Proteinase K Solution described in step 3) is
(15~25): 1.
Preferably, the temperature of the first stage digestion and second stage digestion stands alone as 54~56 DEG C.
Beneficial effects of the present invention: cervical cell lytic reagent box provided by the invention includes lysis buffer and albumen
Enzyme K；Tris-HCl provides buffer system in the lysis buffer；EDTA is able to suppress as divalent metal ion chelator
DNase activity in cell；Surfactant SDS can solubilizing lipids and protein, destroy cell membrane and nuclear membrane structure, make thin
Born of the same parents' rupture, and SDS can also be such that protein and polysaccharide is denaturalized, and make the Separation of Proteins that DNA is coupled, release；NaCl benefit
It is dissolved in DNA；The Proteinase K keeps very high activity in the presence of SDS and EDTA, can become all protein degradations
Polypeptide or small fragment amino acid, make DNA molecular completely be separated.Kit of the present invention has mentioned component
Machine combination can exclude other cells, mucus, drug crystallization, the interference for saving chemical component etc. in liquid, will effectively face
The detected cervical cell saved in liquid of bed uterine neck TCT cracks completely.
Containing in the clinical uterine neck TCT detection process obtained using kit provided by the invention and cleavage method
The cell pyrolysis liquid of cervix cell preservation solution sample carries out routine DNA and extracts, and the DNA concentration of acquisition can achieve 100-
300ng/ μ l, 260/280 value 1.85-2.05,260/230 value 1.5-1.9；After carrying out PCR amplification as template using the DNA of extraction,
The amplification curve of house-keeping gene is good, and CT value is between 25-27.It can be seen that obtained using kit provided by the invention
Lysate carries out DNA extraction, can obtain the DNA of high quality, high concentration, be conducive to the amplification and genetic test of subsequent DNA.
Specific embodiment
Cell cracking is the primary committed step that cell DNA extracts, and whether cell cracking is abundant, and whether DNA release is complete
Entirely, purity and concentration that DNA is extracted directly are determined, the result of subsequent PCR experiment is influenced.Clinical TCT detection saves de- in liquid
It is few to fall cervical cell cell concentration, and it is subsequent to carry out PCR test experience, conventional cell lysing methods are simultaneously not suitable for.The present invention
Using the method for combining chemical action with enzyme effect, the demand to cell concentration can be reduced, and relatively mild, can obtained
Complete DNA chain.
The present invention provides a kind of cervical cell lytic reagent box, the cracking object of the kit is that uterine neck liquid-based is thin
Confluent monolayer cells detection saves the cervical cell in liquid sample；The kit can also be used to the cell of cracking other forms.This
In invention, the kit includes lysis buffer and Proteinase K；The lysis buffer takes water as a solvent, including following dense
The component of degree: the Tris-HCl of 10~500mM, the EDTA of 1~80mM, 0.05~2wt%SDS and 100~250mM NaCl.
In the present invention, the lysis buffer takes water as a solvent, and preferably includes the component of following concentration: 50~
The Tris-HCl of 400mM, the EDTA of 5~60mM, 0.1~1wt%SDS and 150~220mM NaCl；It is furthermore preferred that include with
The component of lower concentration: EDTA, 0.1wt%SDS and 200mM NaCl of Tris-HCl, 50mM of 100mM.
In the present invention, the pH value of Tris-HCl is preferably 8.0~8.3 in the lysis buffer, and more preferably 8.1
~8.2；The effect of the Tris-HCl is to provide buffer system, in the range of so that the pH of whole system is maintained alkalescent；This
EDTA described in invention can inhibit DNase activity in cell as divalent metal ion chelator；SDS is strong denaturant and egg
White lytic agent, detergent can dissolve cell membrane and nuclear membrane, destroy cell, and the Separation of Proteins that DNA is coupled；NaCl
Conducive to DNA dissolution.The present invention is not particularly limited the source of each ingredient in the lysis buffer, using commercial product
?.
The present invention is not particularly limited the source of the Proteinase K, using this field conventional commercial Proteinase K；
In the present invention, the Proteinase K in the kit is preferably commercial protein enzyme Ketamine end or Proteinase K Solution；The Proteinase K
When in use, it is preferably used in the form of Proteinase K Solution；The Proteinase K Solution is preferably molten by solvent of ultrapure water
Proteolytic enzyme Ketamine end obtains.The concentration of heretofore described Proteinase K Solution is preferably 0.1~2mg/ml, more preferably
0.5~1.5mg/ml；The effect of heretofore described Proteinase K is protein degradation matter, separates DNA molecular.The present invention
Described in Proteinase K, very high activity is still able to maintain in the presence of SDS and EDTA, can by all protein degradations become polypeptide or
Small fragment amino acid, makes DNA molecular completely be separated.The enzyme activity of the Proteinase K is preferably 40U/mg.
The present invention also provides the methods using kit cracking cervical cell, comprising the following steps: 1) PBS is slow
Fliud flushing cleans cervical cell sample liquid, is separated by solid-liquid separation, and collects solid phase components；2) solid phase components are mixed with lysis buffer
It closes, obtains mixed liquor；3) mixed liquor is mixed with Proteinase K Solution, carries out first stage digestion, the first stage
The temperature of digestion is 53~58 DEG C, and the time is 10~14h, obtains digestive juice；4) digestive juice is vortexed and shakes 30~60s
Afterwards, continue second stage digestion, the temperature of the second stage digestion is 53~58 DEG C, 1.5~2.5h of time；It is described
Cervical cell sample liquid derives from the clinic detected preservation liquid containing cervical cell of uterine neck TCT.
In the present invention, the cervical cell sample liquid, which derives from clinical uterine neck TCT detection process, contains cervical cell
Preservation liquid.Heretofore described sample liquid directlys adopt the preservation liquid of hospital clinical uterine neck TCT detection without separately preparing
, it is easily obtained.In the present invention, the sample liquid is the preservation liquid containing the cervical cell to fall off；The sample liquid is deposited
It is put under the conditions of -20 DEG C, is solid-state or liquid, the product depending on different company.Heretofore described sample liquid, that is, uterine neck
Complicated component in the detected preservation liquid of TCT, in addition to aim cell (cervical cell), saving can also be there are also non-epithelium in liquid
Cell, acellular components；And the clinical sample obtained is due to the difference in operation of doctor, the difference at cervical lesions position every time
Factors, the cell contents such as property can also have very big otherness.The preservation liquid in clinical sample liquid that the present invention obtains comes respectively
From the product of 2 companies, the Surepath of U.S. company BD and the ThinPrep of Hologic company of the U.S..In above-mentioned preservation liquid
Chemical component containing fixed cell, this kind of chemical component can be such that DNA crosslinks with protein, influence the release of DNA, because
This saves the distracter that liquid itself is also cervical cell DNA release.
The present invention cleans cervical cell sample liquid, solid-liquid with PBS buffer solution after obtaining the cervical cell sample liquid
Solid phase components are collected in separation.In the present invention, the PBS buffer solution and the volume ratio of cervical cell sample liquid are 1~5:1.
The cervical cell sample liquid is uniformly mixed by heretofore described PBS buffer solution cleaning cervical cell with PBS buffer solution.
In the present invention, the mode of the separation of solid and liquid is preferably centrifuged；The centrifugal force of the centrifugation is preferably 4000~5000g, more excellent
It is selected as 4600g, the time of the centrifugation is preferably 50~70min, more preferably 55~65min, most preferably 60min.This hair
It is bright to remove supernatant after the centrifugation, solid phase components are collected, next step operation is carried out.
The solid phase components being collected into are uniformly mixed with lysis buffer and obtain after obtaining the solid phase components by the present invention
Obtain mixed liquor.In the present invention, the volume ratio of the lysis buffer and the cervical cell sample liquid described in step 1) is preferably
(1~5): (8~12), more preferably (2~4): (9~11).The present invention does not have special want to the uniformly mixed method
It asks, in the specific implementation process, the preferred method mixed using pressure-vaccum.
The present invention mixes the mixed liquor with Proteinase K Solution after obtaining the mixed liquor, carries out the first stage
The temperature of digestion, the first stage digestion is 53~58 DEG C, and the time is 10~14h, obtains digestive juice.Institute in the present invention
The volume ratio for stating lysis buffer and Proteinase K is preferably (15~25): 1, more preferably 20:1.Heretofore described first
The temperature of phase digestion is preferably 54~56 DEG C, and more preferably 55 DEG C, the time of the digestion is preferably 11~13h, more preferably
For 12h.
The digestive juice is vortexed after shaking 30~60s after obtaining the digestive juice, continues second by the present invention
The temperature of phase digestion, the second stage digestion is 53~58 DEG C, 1.5~2.5h of time.Heretofore described vortex concussion
Using the vortex concussion instrument of this field routine, the present invention is not particularly limited the frequency for being vortexed concussion, Zhi Nengneng
It is enough to realize the effect mixed.The heretofore described temperature for continuing digestion is preferably 54~56 DEG C, and more preferably 55 DEG C,
The time for continuing digestion is preferably 1.5~2.5h, more preferably 2h.The present invention it is described continue digestion after complete
Cell cracking realizes the complete cracking of cell.
(53~58 DEG C) application surface activating agent SDS solubilizing lipids and protein under the high temperature conditions in the present invention destroy
Cell membrane and nuclear membrane structure make cell rupture, and SDS can also be such that protein and polysaccharide is denaturalized, and make the protein that DNA is coupled
Separation, releases；Proteinase K is added, protein can be hydrolyzed into polypeptide or amino acid, completely isolated conducive to DNA
Come, improves subsequent operation DNA amount to obtain and purity.Heretofore described Proteinase K is below pH4~12.5 and high temperature (50
~70 DEG C) it is active, and Proteinase K enzymatic activity is also unaffected in the presence of EDTA and SDS.Under hot conditions (53~
58 DEG C), the SDS of high concentration, effect and the concussion effect of Proteinase K can be such that cell sufficiently cracks, make DNA and protein
It is more easily separated.Buffer system Tris-HCl provides the environment of certain pH value (alkalescent), is conducive to the stabilization of DNA；Divalent metal
Ion chelating agent EDTA can chelate Mg necessary to DNA enzymatic activity2+、Ca2+, to inhibit DNA enzymatic active, prevent cell broken
DNA enzymatic degradation of dna after broken；NaCl is conducive to the dissolution of DNA.
Technical solution provided by the invention is described in detail below with reference to embodiment, but they cannot be managed
Solution is limiting the scope of the present invention.
Embodiment 1
Cervical epithelial cells lytic reagent box
The preparation of lysis buffer:
Proteinase K: 20mg/ml.
Cell lysing methods:
Isometric PBS cleaning is added in preservation liquid 10ml in clinical uterine neck TCT detection process containing cervical cell,
4600g is centrifuged 60min, carefully removes supernatant, collects precipitating；
The above-mentioned lysis buffer of 1ml is added, piping and druming mixes precipitating, and 50 μ l Proteinase Ks, 55 DEG C of digestion 12h are added；
Be vortexed concussion 50s, and 55 DEG C are continued to digest 2h, until cell cracking is complete.
After cell cracking, DNA is extracted according in paraffin-embedded tissue DNA extraction kit (Beijing Tiangeng/DP331-02)
Step 10 start operation to complete (kit specification), specifically includes the following steps: the liquid subpackage of cracking is arrived
1.5ml EP pipe, every pipe are 220 μ l, and 220 μ l GB are added, is vortexed and mixes, continuously add 250 μ l dehydrated alcohols, is vortexed and mixes
And of short duration centrifugation；The above liquid is added in centrifuge tube CR3 by several times, 12000rpm is centrifuged 1min, abandons liquid；Into centrifugal column
500 μ l GD, 12000rpm are added and are centrifuged 1min, abandon liquid；600 μ l PW, 12000rpm centrifugations are added into centrifugal column
1min abandons liquid；600 μ l PW, 12000rpm are added into centrifugal column again and are centrifuged 1min, abandon liquid；Room temperature of uncapping is dried
5-10min makes dehydrated alcohol sufficiently volatilize；Centrifugal column is put into a new 1.5EP pipe, 20 μ l TE, room temperature 15min are added,
12000rpm is centrifuged 1min, and liquid is rejoined centrifuge tube, and rejoins 40-60 μ l TE, and 12000rpm is centrifuged 1min,
Survey DNA concentration and purity.
PCR system
PCR program:
PCR system and program are as described above
Sample liquid source BD company saves liquid
Sample 1:DNA concentration is 217.0ng/ μ l, 260/280=1.89,260/230=1.87, PCR result house keeper's base
Because CT value is 24.79.
Sample 2:DNA concentration is 153.7ng/ μ l, 260/280=1.91,260/230=1.77, PCR result house keeper's base
Because CT value is 24.66.
Sample 3:DNA concentration is 135.3.0ng/ μ l, 260/280=1.95,260/230=1.67, PCR result house keeper
Gene C T value is 25.15.
Comparative example 1
Samples sources BD company save liquid, sample 1, sample 2 and sample 3 respectively with sample 1, the sample 2 in embodiment 1
It is identical with sample 3.
Cell cracking uses paraffin-embedded tissue DNA extraction kit (Beijing Tiangeng/DP331-02) cell cracking agent
Box, specific method is referring to kit specification.
Sample 1: concentration is 1.5ng/ μ l, 260/280=3.2,260/230=0.05, after can not carrying out because concentration is too low
Continuous PCR experiment.
Sample 2: concentration is 2.2ng/ μ l, 260/280=3.32,260/230=0.03, can not be carried out because concentration is too low
Subsequent PCR experiment.
Sample 3: concentration is 13.5ng/ μ l, 260/280=1.45,260/230=0.04, can not be carried out because concentration is too low
Subsequent PCR experiment.
Embodiment 2
Cervical epithelial cells lytic reagent box
Lysis buffer takes water as a solvent, the component including following concentration: the EDTA of Tris-HCl, 50mM of 200mM,
0.1wt%SDS and 200mMNaCl
Proteinase K: 20mg/ml.
Cell lysing methods:
The PBS cleaning for saving liquid 12ml and 2 times of volumes being added in clinical uterine neck TCT detection process containing cervical cell,
4600g is centrifuged 60min, carefully removes supernatant, collects precipitating；
The above-mentioned lysis buffer of 1ml is added, piping and druming mixes precipitating, and 50 μ l Proteinase Ks, 55 DEG C of digestion 12h are added；
Be vortexed concussion 50s, and 55 DEG C are continued to digest 2h, until cell cracking is complete.
After cell cracking, DNA is extracted according in paraffin-embedded tissue DNA extraction kit (Beijing Tiangeng/DP331-02)
Step 10 start operation to complete (kit specification is detailed), specifically includes the following steps: the liquid subpackage of cracking is arrived
1.5ml EP pipe, every pipe are 220 μ l, and 220 μ l GB are added, is vortexed and mixes, continuously add 250 μ l dehydrated alcohols, is vortexed and mixes
And of short duration centrifugation；The above liquid is added in centrifuge tube CR3 by several times, 12000rpm is centrifuged 1min, abandons liquid；Into centrifugal column
500 μ l GD, 12000rpm are added and are centrifuged 1min, abandon liquid；600 μ l PW, 12000rpm centrifugations are added into centrifugal column
1min abandons liquid；600 μ l PW, 12000rpm are added into centrifugal column again and are centrifuged 1min, abandon liquid；Room temperature of uncapping is dried
5-10min makes dehydrated alcohol sufficiently volatilize；Centrifugal column is put into a new 1.5EP pipe, 20 μ l TE, room temperature 15min are added,
12000rpm is centrifuged 1min, and liquid is rejoined centrifuge tube, and rejoins 40-60 μ l TE, and 12000rpm is centrifuged 1min,
Survey DNA concentration and purity.
The new Bai Shi company in sample liquid source saves liquid
Sample 1: concentration is 63.8ng/ μ l, 260/280=2.00,260/230=1.38, PCR result house-keeping gene CT
Value is 24.2.
Sample 2: concentration is 145.5ng/ μ l, 260/280=1.96,260/230=1.61, PCR result house-keeping gene CT
Value is 23.4.
Sample 3: concentration is 324.7ng/ μ l, 260/280=1.94,260/230=1.72, PCR result house-keeping gene CT
Value is 23.6.
Comparative example 2
The new Bai Shi company in sample liquid source saves liquid
Sample 1, sample 2 and sample 3 respectively in embodiment 2 sample 1, sample 2 and sample 3 it is identical.
Cell cracking uses paraffin-embedded tissue DNA extraction kit (Beijing Tiangeng/DP331-02) cell cracking agent
Box, specific method is referring to kit specification.
Sample 1: concentration: 54.5ng/ μ l, 260/280=1.36,260/230=0.45 can not be carried out because concentration is too low
Subsequent PCR experiment.
Sample 2: concentration: 3.9ng/ μ l, 260/280=0.93,260/230=0.11, after can not being carried out because concentration is too low
Continuous PCR experiment.
Sample 3: concentration: 16.3ng/ μ l, 260/280=1.48,260/230=0.29 can not be carried out because concentration is too low
Subsequent PCR experiment.
As can be seen from the above embodiments, 50~100 μ are being added in the DNA that kit and cleavage method provided by the invention obtain
After l TE, concentration can achieve 100~300ng/ μ l, 260/280 value, 1.85~2.05,260/230 value 1.5~1.9, and PCR expands
After increasing, the amplification curve of house-keeping gene is good, and CT value is between 25~27；And other conventional cleavage methods, using same examination
Agent box extracts, and after 20~40 μ lTE are added, 20ng/ μ l is not achieved in concentration, and 260/280 value range is very big, unstable, 1~3
Between, 260/230 value is between 0~0.1, and concentration and purity is too low can not carry out subsequent PCR experiment can not carry out PCR knot
The comparison of fruit.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications
It should be regarded as protection scope of the present invention.
Claims (10)
1. a kind of cervical cell lytic reagent box, which is characterized in that including lysis buffer and Proteinase K；The cracking buffering
Liquid takes water as a solvent, the component including following concentration: the Tris-HCl of 10~500mM, the EDTA of 1~80mM, 0.05~2wt%
SDS and 100~250mM NaCl.
2. kit according to claim 1, which is characterized in that the component including following concentration: 50~400mM's
Tris-HCl, the EDTA of 5~60mM, 0.1~1wt%SDS and 150~220mM NaCl.
3. kit according to claim 1, which is characterized in that the lysis buffer includes the component of following concentration:
EDTA, 0.1wt%SDS and 200mM NaCl of Tris-HCl, 50mM of 100mM.
4. kit according to claim 1, which is characterized in that the pH value of the Tris-HCl is 8.0~8.3.
5. utilizing the method for the cracking cervical cell of kit described in 4 any one of Claims 1 to 4, comprising the following steps:
1) PBS buffer solution cleans cervical cell sample liquid, is separated by solid-liquid separation, and collects solid phase components；
2) solid phase components are mixed with lysis buffer, obtains mixed liquor；
3) mixed liquor is mixed with Proteinase K Solution, carries out first stage digestion, the temperature of the first stage digestion is
53~58 DEG C, the time is 10~14h, obtains digestive juice；
4) digestive juice is vortexed after shaking 30~60s, continues second stage digestion, the temperature of the second stage digestion
Degree is 53~58 DEG C, 1.5~2.5h of time；
The cervical cell sample liquid derives from the clinic detected preservation liquid containing cervical cell of uterine neck TCT.
6. according to the method described in claim 5, it is characterized in that, the volume of the PBS buffer solution and cervical cell sample liquid
Than for 1~5:1.
7. according to the method described in claim 5, it is characterized in that, lysis buffer described in step 2) and institute in step 1)
The volume ratio for the cervical cell sample liquid stated is (1~5): (8~12).
8. according to the method described in claim 5, it is characterized in that, the concentration of the Proteinase K Solution is 0.1~2mg/ml.
9. according to the method described in claim 8, it is characterized in that, lysis buffer described in step 2) and institute in step 3)
The volume ratio for the Proteinase K Solution stated is (15~25): 1.
10. according to the method described in claim 6, it is characterized in that, the temperature of first stage digestion and second stage digestion
Degree stands alone as 54~56 DEG C.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110373410A (en) * | 2019-06-12 | 2019-10-25 | 江苏莱尔生物医药科技有限公司 | A kind of nucleic acid rapid cleavage liquid and preparation process and its application method |
| CN112626174A (en) * | 2020-12-16 | 2021-04-09 | 广州源井生物科技有限公司 | Cell lysis solution and application thereof |
| CN113957122A (en) * | 2020-07-21 | 2022-01-21 | 厦门希吉亚生物科技有限公司 | Method for extracting DNA from cervical cell sample |
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| CN110373410A (en) * | 2019-06-12 | 2019-10-25 | 江苏莱尔生物医药科技有限公司 | A kind of nucleic acid rapid cleavage liquid and preparation process and its application method |
| CN113957122A (en) * | 2020-07-21 | 2022-01-21 | 厦门希吉亚生物科技有限公司 | Method for extracting DNA from cervical cell sample |
| CN113957122B (en) * | 2020-07-21 | 2024-01-26 | 厦门赛尔吉亚医学检验所有限公司 | Method for extracting DNA from cervical cell sample |
| CN112626174A (en) * | 2020-12-16 | 2021-04-09 | 广州源井生物科技有限公司 | Cell lysis solution and application thereof |
| CN112626174B (en) * | 2020-12-16 | 2024-05-07 | 广州源井生物科技有限公司 | Cell lysate and application thereof |
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